1. Field of the Invention
This invention relates to a method for preparing an analytical element useful for immunoassay utilizing antigen-antibody reaction.
2. Description of the Prior Art
Recently, an analytical method using a dry-type analytical element has been developed. In a dry-type analytical element, generally, an analyte such as a biochemical substance contained in a body fluid is allowed to react with reagent(s) incorporated in the analytical element. The amount of a particular reaction product or unreacted component is then determined by measuring the coloring, discoloring, fluorescence, emission or the like, optically such as by spectrophotometry, to determine the content of the analyte. By using the dry-type analytical element, a particular component such as a biochemically active substance in a liquid sample can be analyzed simply, rapidly and accurately.
On the other hand, an immunoassay utilizing a dry-type analytical element has been reported in U.S. Pat. No. 4,459,358, Japanese Patent KOKAI 49-53888 and 59-102388.
The antigen-antibody reaction in the field of immunology is the reaction in which an antibody or antigen specifically reacts with the corresponding antigen or antibody alone. It is widely utilized for the diagnosis of diseases of the autoimmune system, detection of a trace component in a living body and the like. However, since methods utilizing an antigen-antibody reaction requires significant operational skills, it has been desired in the field of clinical tests to develop a measuring method requiring only simple operations which produces accurate results.
The method of measuring an antigen disclosed in Japanese Patent KOKAI 59-77356 (1984) is a typical method utilizing a multilayer analytical element. This analytical element is composed of a spreading layer containing an antigen labeled with a fluorescent material, a partition layer composed of a porous medium and a reaction layer containing an immobilized antibody. The amount of the antigen is determined in this element by measuring the decrease of fluorescence intensity, utilizing the competitive antigen-antibody reactions between the antigen in a sample solution and the labeled antigen.
As another immunoassay method having a relatively high sensitivity, enzyme immunoassay (EIA) is known. A typical EIA is the competitive reaction solid phase method comprising allowing the antigen to be measured and an enzyme-labeled antigen or its derivative to react competitively with immobilized antibody, conducting bound-free (B/F) separation, and measuring the activity of either the enzyme bound to the antibody or the free enzyme to determine the amount of the antigen to be measured. In order to eliminate, the B/F separation, the enzyme whose activity increases or decreases by binding to the antibody is necessary.
In order for these measuring systems to work effectively, two components, i.e. the enzyme-labeled antigen or its derivative and the immobilized antibody in one case, or enzyme labeled antibody or its derivative and immobilized antigen in the other case, are necessary. In either case, these two components should be separated so as to not react with each other before measuring. Satisfactory analytical sensitivity was not obtained in most of the known dry-type analytical elements because of a low signal/background ratio caused by insufficient separation of the two components.